Difference between revisions of "Team:Paris Saclay/Experiments"

(→‎Protocols)
(→‎Protocols)
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Centrifuge at 13000rpm for 1min to pellet the cells.  
 
Centrifuge at 13000rpm for 1min to pellet the cells.  
 
Discard supernatant and resuspend cells in 100µL of TE buffer.  
 
Discard supernatant and resuspend cells in 100µL of TE buffer.  
−
Add 200µL of solution II and mix gently by inverting the tubes until lysates appears clear. (dissolution), then 150µL of solution III is added and mixed them by inverting the tubes (precipitation). The solutions are kept onto ice for 10min. Then they are centrifuged 10min at 13000rpm. The supernatant is recovered. 100µL of phenol is added in each tubes (denature proteins) and vortexed 30seconds. After, the tubes are centrifuged 7min at 13000rpm. Aqueous phase is recovered (which contains DNA). 2 volumes (900µL) of 100% ethanol are added and solutions are put into -20°C for 10min. Then, the tubes are centrifuged 10min at 13000rpm. Supernatants are discarded and 800µL of 70% ethanol is added to remove ions making sure the pellet containing DNA remains at the tube bottoms. The tubes are centrifuged 4min at 13000rpm and supernatants are removed. The tubes are dried in speedvac. The pellet is resuspended in 50µL of TE/RNAse. The tubes are kept at -20°C.
+
Add 200µL of solution II and mix gently by inverting the tubes until lysate appears clear.
 +
Add 150µL of solution III and mixed gently.
 +
Keep the solution on ice for 10min.  
 +
Centrifuge for 10min at 13000rpm and recover the supernatant.
 +
Add 100µL of phenol in each tubes to denature the proteins and vortex for 30s.  
 +
Centrifuge the tubes for 7min at 13000rpm.  
 +
Recover the aqueous phase.
 +
Add 2 volumes (900µL) of 100% ethanol and put at -20°C for 10min.  
 +
Centrifuge the tubes again for 10min at 13000rpm.  
 +
Discard supernatant and wash with 800µL of 70% ethanol to remove the remaining ions. Make sure that the pellet containing DNA remains at the bottom of the tubes.  
 +
Centrifuge the tubes for 4min at 13000rpm and remove supernatants.
 +
Dry tubes in speedvac.  
 +
Resuspend the pellet in 50µL of TE/RNAse.  
 +
Keep the extracted plasmids at -20°C.
  
 
'''TE buffer recipe'''
 
'''TE buffer recipe'''
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|55mM
 
|55mM
 
|}
 
|}
−
Dissolve HEPES, CaCl<sub>2</sub> and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl<sub>2</sub>. Filter to sterilize and keep at 4°C.
+
 
 +
'''Solution II recipe (lysis solution)''' Keep for maximum 3 weeks.
 +
{| class="wikitable"
 +
|-
 +
|SDS
 +
|1%
 +
|-
 +
|NaOH
 +
|0.1M
 +
|}
 +
 
 +
 
 +
'''Solution III recipe (neutralization solution)''' CH<sub>3</sub>COOK 3M at pH4.8
 +
{| class="wikitable"
 +
|-
 +
|CH<sub>3</sub>COOK 5M
 +
|60mL
 +
|-
 +
|CH<sub>3</sub>COOH
 +
|11.5mL
 +
|-
 +
|water
 +
|28.5mL
 +
|}
 +
 
 +
'''TE/RNase''' Use only fresh solution.
 +
{| class="wikitable"
 +
|-
 +
|RNase (10mg/mL)
 +
|5µL
 +
|-
 +
|TE
 +
|1mL
 +
|}

Revision as of 14:21, 7 July 2016

Experiments

Describe the experiments, research and protocols you used in your iGEM project.

What should this page contain?

  • Protocols
  • Experiments
  • Documentation of the development of your project

Protocols

Heat shock competent cells

Preparation

Day 1 Inoculate cells in 3.5mL LB medium. Incubate at 37°C overnight.

Day 2 Measure culture OD at 600nm. Dilute cells in 250mL of LB medium so OD equals to 0.12. Incubate overnight at 20°C and 180rpm.

Day 3 Measure culture OD at 600nm and dilute to obtain OD600nm=0.6. Cells must be kept at 4°C during all following steps. Put on ice for 10min and centrifuge for 10min at 3000rpm and 4°C. Discard supernatant and resuspend cells in 80mL of fresh TB buffer. Keep on ice for 10min and centrifuge for 10min at 3000rpm and 4°C. Discard supernatant again and resuspend cells in 20mL of fresh TB buffer with 7% of DMSO. Keep on ice for 10min. Aliquot cells and freeze with liquid nitrogen. Keep at -80°C.

TB buffer recipe

HEPES 10mM
MnCl2 55mM
CaCl2 15mM
KCl 250mM
KOH

Dissolve HEPES, CaCl2 and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl2. Filter to sterilize and keep at 4°C.

Tranformation

Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid). Keep tubes on ice for 30min and heat shock at 42°C for 1min. Add 500µL of LB medium into each tube and incubate at 37°C for 1h. Spread cells on Petri dishes in duplicate and incubate at 37°C overnight.


Electro-competent cells

Preparation

Inoculate 15mL of LB with 200µL of an overnight cell culture. Incubate at 37°C and 180rpm until OD600nm reaches 0.6. Centrifuge cells for 10 minutes at 4000rpm. Wash twice with 10mL of 10% glycerol. Put cells in 200µL of 10% glycerol and use for electroporation.

Transformation

Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid). Electroporate (the variable TODO??? should be close to 6) and recover cells with 1mL of cold LB medium. Incubate cells for 1h at 37°C. Spread cells on Petri dishes in duplicate and incubate at 37°C overnight.


Plasmid DNA extraction

Transfer 1.5mL of an overnight culture into an eppendorf tube. Centrifuge at 13000rpm for 1min to pellet the cells. Discard supernatant and resuspend cells in 100µL of TE buffer. Add 200µL of solution II and mix gently by inverting the tubes until lysate appears clear. Add 150µL of solution III and mixed gently. Keep the solution on ice for 10min. Centrifuge for 10min at 13000rpm and recover the supernatant. Add 100µL of phenol in each tubes to denature the proteins and vortex for 30s. Centrifuge the tubes for 7min at 13000rpm. Recover the aqueous phase. Add 2 volumes (900µL) of 100% ethanol and put at -20°C for 10min. Centrifuge the tubes again for 10min at 13000rpm. Discard supernatant and wash with 800µL of 70% ethanol to remove the remaining ions. Make sure that the pellet containing DNA remains at the bottom of the tubes. Centrifuge the tubes for 4min at 13000rpm and remove supernatants. Dry tubes in speedvac. Resuspend the pellet in 50µL of TE/RNAse. Keep the extracted plasmids at -20°C.

TE buffer recipe

Tris 10mM
EDTA 55mM

Solution II recipe (lysis solution) Keep for maximum 3 weeks.

SDS 1%
NaOH 0.1M


Solution III recipe (neutralization solution) CH3COOK 3M at pH4.8

CH3COOK 5M 60mL
CH3COOH 11.5mL
water 28.5mL

TE/RNase Use only fresh solution.

RNase (10mg/mL) 5µL
TE 1mL